The T cell molecule CTLA-4 plays an important role in downregulating T cell immune responses. Mice deficient in CTLA-4 die at several weeks of age with massive polyclonal T cell expansion and fulminant autoimmunity. The inhibitory signals by which CTLA-4 regulates T cell responses remain incompletely understood. CTLA-4, which can be tyrosine-phosphorylated, has two cytoplasmic Tyr residues (positions 165 and 182), both of which are conserved in the related molecule, CD28. These two conserved Tyr are phosphorylation sites in CD28, and each has been implicated in the delivery of the CD28 costimulus. We propose that interactions with Tyr 165 and 182 are critical for the inhibitory function of CTLA-4. To address these possibilities we will use gene targeting to produce mice with Phe for Tyr mutations at either position 165 or 182 of the ctla-4 gene. We will apply recently developed strategies to introduce site specific mutations in the mouse genome. To this end we will subclone isogenic genomic fragments of ctla-4 into an insertional targeting vector containing positive and negative selection and use the "hit and run" strategy to introduce desired Phe for Tyr substitution mutations into exon 4 of ctla-4 in ES cell lines. ES subclones containing the desired mutations will be used to create chimeric mice carrying the desired mutation, which will then be used to establish heterozygotes and finally homozygotes. The dramatic phenotype of the CTLA-4-/- mice will facilitate analysis of the consequences of these mutations.